Several compounds can prevent carcinogen-induced tumor formation in experimental animals, but little is known about their mechanism of action. One approach is to test their inhibitory effect on the carcinogen-induced transformation of mammalian cells in culture since this allows dissection of the process into various stages which can be critically examined in a way not possible with whole animals. Several investigators have shown inhibition in 10T1/2 cells or hamster embryo cells, but the mechanisms of action of the agents are not yet well-defined. There is little information on the interaction of anticarcinogenic agents with human cells in culture and almost no information on their effects of transformation of human cells. We have a reproducible quantitative assay for carcinogen-induced neoplastic transformation of diploid human fibroblasts. The assay can be divided into two parts: (1) induction of anchorage independence (AI) and (2) production of tumors in athymic mice. Part One consists of exposing the cells to a carcinogen during exponential growth, allowing 8 cell divisions, assaying the progeny for ability to form colonies in soft agar. AI cells form colonies after 4 weeks. Part Two consists of isolating theses AI cells, propagating them, and injecting their progeny subcutaneously into sub-lethally X-irradiated athymic mice. Tumors (fibrosarcomas) arise at the site of inection within 10 days 90-100% of the time. We will use this assay to determine the effectiveness of selected representatives of the following categories of carcinogens: (1) Antioxidants, (2) Ascorbic Acid, (3) Retinoids, (4) Protease Inhibitors (5) Steroids and (6) Miscellaneous compounds. Each agent will be assayed for ability to inhibit induction of anchorage independence and/or production of tumors. The agents which prove positive will be further tested to determine which specific substages it affects, e.g., (1) interference with DNA binding; (2) inhibition of metabolic activation of parent carcinogens; (3) lengthening time available for DNA repair or increasing the rate; (4) blocking formation or agar colonies; (5) preventing induction of tumorigenic cells; and (6) preventing tumor formation. When the stage(s) is known, we will attempt to determine the mechanism of action. Since the induction of AI in human cells resembles a somatic cell mutation, the agents which prove able to interfere with AI induction will also be tested for ability to inhibit carcinogen-induced mutations to TG or DT resistance.